Device and method for the examination of samples in a non vacuum environment using a scanning electron microscope

ABSTRACT

A chamber suitable for use with a scanning electron microscope. The chamber comprises at least one aperture sealed with a membrane. The membrane is adapted to withstand a vacuum, and is transparent to electrons and the interior of the chamber is isolated from said vacuum. The chamber is useful for allowing wet samples including living cells to be viewed under an electron microscope.

CROSS-REFERENCE TO PRIOR APPLICATION

This application is a continuation of U.S. patent application Ser. No.10/448,313 May 27, 2003, now U.S. Pat. No. 6,992,300, which is acontinuation of International Application No. PCT/IL01/01108, filed Nov.30, 2001, which claims the benefit of Provisional Application No.60/250,879, filed on Dec. 1, 2000 and Provisional Application No.60/306,458, filed on Jul. 20, 2001, each of which are incorporated byreference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to a device and method for the examinationof samples in a non-vacuum environment using a scanning electronmicroscope and more particularly but not exclusively to the use of suchan apparatus and method for inspection of objects in a wet environment.

BACKGROUND OF THE INVENTION

Optical microscopy is limited, by the wavelength of light, toresolutions in the range of a hundred, and usually hundreds, ofnanometer. Scanning electron microscopes (SEMs) do not have thisimitation and are able to attain a considerably higher resolution, inthe range of a few nanometers.

One of the disadvantages of SEMs is that samples have to be maintainedin vacuum, precluding the study of in-vivo processes or the study of wetmaterials. Furthermore, electrically insulating samples composed oforganic materials require coating to avoid charge accumulation.

As early as 1960 a thesis by Thornley (University of Cambridge, 1960)disclosed unsuccessful attempts to maintain a sample intended forelectron microscopy in an atmosphere of water vapor. A membrane is usedto seal a chamber from the vacuum of the electron beam and the chamberitself has an inlet from a source of water vapor.

Attempts to use Electron Microscopy for living specimens go back as faras 1970. A paper by Swift and Brown (J. Phys. E: Sci. Instrum. 3, 924,1970) disclosed the use of transmission electron microscopy (TEM) forexamination of specimens at atmospheric pressure, for example in water.A cell having an aperture sealed with a collodion-carbon film is used tomount a sample. An electron beam passes through the aperture to strikethe sample, and electrons not stopped by the sample continue to ascintillator where photons are produced. At atmospheric pressure theresults were found to be “rather noisy” although a resolution of 0.1 μmwas claimed.

U.S. Pat. No. 4,071,766 describes an attempt to use electron microscopyto see material in a non-vacuum environment and refers to the inspectionof living objects as “an ever-recurring problem”. U.S. Pat. No.4,720,633 describes a further attempt to use election microscopy to seematerial in a non-vacuum environment. In both of these patents theelectron beam travels through an aperture to a wet specimen. Neither ofthese attempts succeeds, however, in successfully viewing wet objects.The contents of both of these documents are hereby incorporated byreference.

A commercial product which attempts to solve the above problem is anEnvironmental Scanning Electron Microscope (ESEM), commerciallyavailable from Philips Electron Optics of Eindhoven. The Netherlands,which maintains a vacuum gradient along the electron beam path. However,the ESEM requires working with a sample at a critical point ofwaiter-vapor equilibrium, and requires cooling of the sample to around4° C. Inspection of specimens at pressures of up to 5 Torr is said to bepossible. However, so far there is no evidence that wet and/or livingobjects can be viewed at resolutions of 10 nm and below. Furtherinformation on this product and how it works can be found in U.S. Pat.Nos. 5,250,308, 5,362,964, and 5,412,211, the contents of which arehereby incorporated by reference.

A common method of achieving high resolution inspection of organicmatter is Transmission Electron Microscopy (TEM). TEM requires speciallyprepared specimens having typical thicknesses in the range of 50 nm. Avery high voltage is applied to create a parallel beam that passesthrough the sample. U.S. Pat. No. 5,406,087, the contents of which arehereby incorporated by reference, discloses a specimen holding devicefor use with a TEM. A specimen is sealed between two films that are ableto transmit an electron beam. The interior of the device is filled withmoisture and may be placed within the vacuum environment of the TEM. Avery high energy beam travels through the specimen and surrounding fluidleading to a poor signal to noise ratio, as well as considerable damageto the sample.

The information made available by EM is usually unavailable by othertechniques (reviewed in Griffiths (2001) Trends in Cell Biology,11:4:153-154). The main reason for the prevalent underutilization of EMis the complexity of sample preparation that is not only labor intensiveand time consuming, but also raises concerns regarding the biologicalrelevance of the results. The ability to carry out EM in an aqueousenvironment would obviate these problematic sample preparation steps.

At present, therefore, despite a long-felt need, there is no microscopethat permits the study of wet samples at resolutions showingmacro-molecular and molecular levels of detail. Such all ability isneeded in fields as diverse as cell biology and polymer science is wellis industries such as petroleum, food and microelectronics. Inparticular in the field of cell biology, such a microscope would enableanalysis of cells leading to measurements of molecular level processesand also opening a whole new field in pharmaceutical drug discovery anddiagnostic measurement. For example, such a microscope would allowdetailed study and direct observation of interactions between drugs andliving cells.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a chamber, adaptedfor use with a scanning electron microscope, that will enable electronmicroscopy of wet samples.

In accordance with a first aspect of the present invention there isprovided a chamber suitable for use with a scanning electron microscopesaid chamber comprising at least one aperture, which aperture is sealedwith a membrane, said membrane being adapted to withstand a vacuum, andbeing transparent to electrons and wherein said clamber is isolated fromsaid vacuum.

Preferably, the chamber is adapted to hold water or any aqueous medium,including but not limited to cell culture medium, at substantiallyatmospheric pressure.

Preferably, the aperture sealed by the membrane, has a diameter withinthe range of 0.1 to 4 mm.

Preferably, the membrane has a thickness lying substantially within therange of 200-5000 Å, more preferably between the range of 500 and 2000Å, and most preferably between the range of 500 and 1500 Å.

The properties required of the membrane, which constitutes a mainelement of the present invention, include the ability to withstand apressure differential of approximately one atmosphere between the vacuumof the electron microscope and the environment contained within thechamber sealed by said membrane, while it is thin enough for energeticelectrons to pass through and interact with the sample provided withinthe chamber. Any material with these attributes may be suitable for usein accordance with the principles of the present invention. According tocurrently preferred embodiments, the membrane material is selected fromthe group consisting oft polyimide, polyamide, polyamide-imide,polyethylene, polypyrrole and additional conducting polymers, parlodion,Collodion, Kapton, FormVar, Vinylec, ButVar, Pioloform, silicon dioxide,silicon monoxide, and carbon.

Preferably, a sample is placed in proximity to the membrane.Alternatively a sample is mounted in contact with said membrane and thisgives the advantage that the electron beam has to traverse a minimaldistance through matter (as opposed to vacuum) to reach the sample. Themembrane is preferably supported by a supporting grid placed between themembrane and a vacuum.

In general, the chamber is located in a vacuum whilst enclosing a samplewithin a fluid or at substantially atmospheric pressure or both. Atleast part of a wall of said chamber is resistant to a one atmospherepressure gradient and is transparent to electrons having an energygreater than around 2 keV.

According to a second aspect of the present invention there is providedapparatus for high resolution inspection of an object in a chamber usinga scanning electron beam, comprising a vacuum path for the electron beamand a vacuum-resistant barrier placed in association with the object,said vacuum resistant barrier being placed such as to isolate the objectfrom the vacuum. The object may have thereon a fluorescent and anelectro-luminescent marker, whereby a beam of electrons are able toexcite photo-emission. The marker may be associated with a particularmolecule of interest or the like and this method allows for light basedimages containing information about individual molecules. Such anembodiment preferably comprises an optical sensing unit and the chambermay be built as part of a light guide. The optical sensing unit may alsocomprise a photo-multiplier tube.

The optical sensing unit may be adapted to sense individual photons, andthe apparatus is thereby adapted to sense light emissions resulting fromthe stimulation of single molecules.

The barrier or membrane may, in a more preferred embodiment, be any oneof polyimide, polyamide, polyamide-imide, Kapton, FormVar and ButVar.

The barrier or membrane may be reinforced with a reinforcing grid placedbetween said membrane and said vacuum, and may have a thickness lyingwithin a range of substantially 200 to 5000 Å, more preferably in therange of 500 to 2000 Å, most preferably in the range of 500-1500 Å.

Preferably, the barrier or membrane is placed across an aperture havinga diameter substantially within the range of 0.1 to 4 mm. In aparticularly preferred embodiment the diameter is substantially 1 mm.

The barrier or membrane is preferably transparent to energeticelectrons, that is to say electrons having energies in excess of 2 keVor 3 keV.

According to one embodiment, the barrier is coated with a layer of amaterial having a high secondary electron yield, typically lithiumfluoride.

In one embodiment, radical scavenger molecules are located inassociation with said object.

In one embodiment the object or sample is placed adjacent to or incontact with said vacuum-resistant barrier.

According to a third aspect of the present invention there is providedthe use of a membrane, of which an exposed area having a diameter of 1mm is resistant to a one-atmosphere pressure gradient, and which istransparent to high energy electrons, to construct a chamber to enablewet samples to be viewed under a scanning electron beam at a resolutionsubstantially better than 100 nm.

According to a fourth aspect of the present invention there is provideda method of observing wet objects at nanometer range resolutioncomprising the steps of isolating a wet object in a chamber byseparating said chamber from a vacuum chamber using a barrier, providingan electron beam to strike said sample from said vacuum chamber throughsaid barrier, and observing at least one of secondary and backscatteredelectrons emerging from said chamber.

Preferably, barrier comprises a membrane selected to withstand apressure of substantially one atmosphere arid to be transparent toelectrons having an energy in excess of around 2 keV.

Preferably, the wet object comprises a pharmaceutical composition.

Preferably, the wet object further comprises a living cell with whichthe pharmaceutical composition is interacting.

Preferably, said chamber comprises a fluid inlet and wherein thepharmaceutical composition is changed in respect of one of a groupcomprising concentration and type through the inlet dynamically duringthe observation.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the invention and to show how the same maybe carried into effect, reference is now made, purely by way of example,to the accompanying drawings, in which:

FIG. 1 is a generalized diagram showing a longitudinal cross-section ofthe sample area of a prior art scanning electron microscope.

FIG. 2 is a generalized diagram showing a longitudinal cross-section ofa scanning electron microscope incorporating a chamber according to anembodiment of the present invention.

FIG. 3 is a generalized cross-sectional diagram of a chamber accordingto one embodiment of the present invention.

FIG. 4 is a generalized cross-sectional diagram of a chamber accordingto another embodiment of the present invention, embedded within a lightguide.

FIG. 5 is an electron micrograph taken using the embodiment of FIG. 3.

FIG. 6 is a diagram of an assembled sample chamber unit forfluorescence.

FIG. 7 containing FIGS. 7A, 7B, 7C/1, 7C/2, 7D, 7E/1 and 7E/2 depictsdiagrams of components A-E of the chamber of FIG. 6.

FIG. 8 including FIGS. 8A and 8B is a diagram of the microscope stageadapter for use with the chamber.

FIG. 9 is a diagram of an alternate embodiment for luminescence.

FIG. 10 including FIGS. 10A-E shows images of fixed wet cells under thepartition membrane.

FIG. 11 including FIGS. 11 A-D show images of live non-labeled adherentcells under the membrane.

FIG. 12 is an image of intact unlabeled CHO cells under the membrane.

FIG. 13 an image of live non-adherent cells under the membrane.

FIG. 14 including FIGS. 14A-C shows cathodoluminescence images of fixedNIH3T3 cells.

FIG. 15 is an image of intact NIH3T3 cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a new technique of scanning electronmicroscopy that is adapted to the study of wet samples. The wetenvironment is provided in a small chamber enclosed by a membrane thatis thin enough for energetic electrons to pass through and interact withthe sample being studied.

We now disclose in detail both the technique and the mechanisms ofsignal formation in the imaging of samples in a scanning electronmicroscope through a membrane. We further disclose the mechanical andspatial setup and the properties required of the membrane, a mainelement in this method.

We disclose simple measurements for the characterization of themembrane, giving a guide to the choice of material and thickness. Wefurther disclose the capabilities of the technique in imaging a varietyof different samples. We evaluate the accessible contrast andresolution, and the current needed to obtain them. We show that it ispossible to observe gold particles in water with a resolution on theorder of 10 nm, while lower contrast materials may give significantlylower resolution on the order of 100 nm. This new technique may beadapted to numerous applications in diverse fields such as materialsresearch and cell biology.

The present invention is based on the isolation of the fluid sample fromthe vacuum by the introduction of a membranous partition. Recentdevelopments in polymer technology enable the production of thinmembranes that are practically transparent to energetic electrons, yetare tough enough to withstand atmospheric pressures on one side and highvacuum on the other side. The imaged volume is within close proximity ofthe membrane, typically a few μm into the fluid. This is ideal for theinspection of objects that are at or adjacent to the membrane surfacesuch as adherent biological cells. The techniques disclosed arefurthermore easily adaptable to all existing Scanning ElectronMicroscopes, enabling measurements at room temperature and atatmospheric pressure.

A. The Experimental Setup

We have successfully applied the devices and methods of the presentinvention to work with both a JEOL 6400 SEM and a Philips ESEM in highvacuum mode, but the devices we present can be used with any SEM.Certain currently preferred embodiments or the present invention will bedescribed below in full detail in relation to the accompanying drawingsand figures.

The sample is held inside a chamber that is designed along the lines ofa standard SEM sample mount insert. The main emphasis is on sealing ofsample against the external pressure difference, and this is done with aseries of o-rings. Closing the sample presents a hazard to the membrane,since considerable deformation would be caused if one were to try andcompress the fluid inside the chamber as we close the sample with themembrane in place. To avoid this a small release channel is kept openthrough the stem of the insert, equilibrating the pressure inside by aslight release of fluid. The opening to this channel is subsequentlysealed off with another o-ring that is screwed in place.

The membrane must have several important properties. First, it needs tobe as transparent to electrons as possible. This implicates a lowaverage atomic number (low Z) and a low density. Polymer films aretherefore the most adequate choice.

Very good mechanical properties are also required of the membrane. Whilekeeping thickness as small as possible to minimize scattering of theelectrons before they reach the zone out interest, the membrane mustresist a difference of pressure of one atmosphere for as large a surfacearea as may be required for observation. It must also be flexible enoughto enable a considerable amount of handling in preparation of thesample, ruling out carbon films since they are very brittle. Theporosity to the materials comprising the sample holder and inside thesample must be reduced as much as possible to ensure proper sealing ofthe chamber.

Ideally, the electrical conductivity of the membrane should be highenough to prevent the local charging of the external surface of themembrane, which may perturb the incident electron beam and blur theimage. In practice we evaporate a thin carbon film on the outside of themembrane, thus eliminating charging effects.

Finally, the affinity of the membrane for the object observed may be animportant factor. Typically in electron microscopes, the electrons gofrom the upper region to the bottom, so that the objects we would liketo observe are located below the membrane. In the method developed, thebest results are obtained when the objects are in close proximity to themembrane, and best when they are attached to it.

Different materials have been tested to build the membrane, all based onCarbon compounds. Formvar anti Butvar, commonly used in TEM to buildsupporting films [Davison and Colquhoun, J. Elec. Microsc. Tech. 2, 35,1985; Handley and Olsen, Ultramicroscopy 4, 479, 1979], and Polyimidehave been tested. Of these materials tested to date only the last fullymet all mechanical and seating requirements. According to one currentlypreferred embodiment we use polyimide membranes of 1450 Å thicknesssupplied by Moxtex, Inc. Polyimide membrane show no measurable porosityto water samples are placed in a vacuum of approximately 10⁻⁷ barr forseveral hours prior to insertion into the SEM, without detectable loss.They can resist the forces produced by atmospheric pressure in windowsover 1 mm². To minimize this force on the membrane and subsequent riskof rupture, the surface was usually reduced by the use of a TEM Ni gridthat was attached to the external side of the membrane (125 to 330 μmmesh were used). For electrical conductivity, a carbon deposition of 50Å on the external surface was found to be sufficient. Affinityproperties, if required, are achieved by internal surface treatments.

B. Mechanism of Contrast Formation

There are two different contributions to the formation of a signal onthe detector. The first is a source of uniform noise, while the secondincludes the signal:

1. When the beam nits the membrane, backscattered electrons (BSE) andsecondary electrons (SE) are produced by the membrane itself. Only theSE produced in the first few nanometers (the mean free path ofsecondaries), can escape from the membrane [Goldstein et al., ScanningElectron Microscopy and X-ray Microanalysis, pp. 113, Plenum press, NewYork, London, 1992]. Their mean free path is around 1 nm in conductors(the thin carbon layer) and up to 10 nm in insulators (as are Polyimideand the fluid beyond it). The SE are created both in the membrane and inthe carbon coating shield.

We therefore get a first contribution to the signal due to the membrane,comprised of both BSE and SE. This contribution is homogeneous sinceboth the composition and the thickness of the membranes we use are thesame everywhere. In the following, the subscript ‘m’ (for membrane) willrefer to this contribution.

2. The portion of electrons from the beam which is not backscatteredwhen crossing the membrane impinges upon the sample. Again, secondaryelectrons and backscattered electrons are produced.

The SEs produced here have no chance to escape, they would get stoppedby either the membrane (an insulator) or the thin carbon coating shield(conductor). In contrast, BSE created in the sample have the possibilityto exit back out through the membrane. As they cross the membrane, theymay generate secondary electrons, which, if created at a distance fromthe surface which is below their mean free path, can escape to thedetector.

Thus, we get this second contribution to the signal due tobackscattering events in the zone of interest, which reach both the SEand BSE detectors. In the following, the subscript ‘s’ (for sample) willrefer to this contribution.

The second contribution (s) is obviously the contribution of interest.It carries the information we seek while the first is related to themembrane only (m). A, contrast between two neighboring points will beobservable if the difference in the sample signal between them is higherthan the fluctuations in the membrane signal.

Our ability to form an image is thus determined by the possibility toobtain a large signal to noise ratio. This in turn is determined by thedifference in backscattering coefficients of the materials locatedinside the sample (“material contrast”), and depends on the membrane aswell.

Given two different materials for which the backscattering coefficientsare known and for a specific membrane, we would like to determine theconditions we need to fulfill in order to form an image.

The total signal collected S is composed of both secondary electrons andbackscattered electrons:S−ε_(BS)η÷ε_(SE)δ  (1)

η and δ represent respectively the ratios of BSE and SE currents to thebeam current. The coefficient ε represents the collection efficiency forthe two kinds of electrons that are detected.

The backscattering coefficient η is on its own made of twocontributions: BSE from inside the membrane (m) and BSE from inside thesample (s), η=ηm+ηs.

Similarly, the SE scattering coefficient δ has two contributions,δ=δm÷ηs Δm.

The difference is that while δm represents the secondary electronsgenerated by electrons of the beam entering the membrane, Δm representsthe secondaries generated in the membrane by electrons on their way out,after a relevant backscattering event inside the sample.

The backscattering coefficient ηs thus multiplies Δm, because the fluxof energetic beam electrons traveling back through the membrane isreduced from unity by ηs.

Note that δm and Δm both describe emission of secondaries from close tothe surface of the membrane. For Δm, the high energy electronsgenerating the secondaries are for the main part the electrons of theincoming beam, with a small contribution from electrons that havebackscattered inside the membrane. For Δm, the electrons generating thesecondaries have experienced a backscattering event inside the sample.For this reason. Δm can contain a slow material dependence. Depending onthe material located below the membrane, the energy spectra of the BSEin this region can be different, and different energies of BSE maygenerate different SE emissions.

Note that the definitions of backscattering and secondary coefficientswe use and the coefficients usually described in the literature differslightly. The latter are defied for a semi-infinite medium and arecharacteristic of the material (Gold, Carbon, Nylon, etc.). Here, thecoefficients describe the charge emission for a membrane with itsparticular thickness and its carbon shield. They do not describe thecharge emission of a semi-infinite sample of polyimide material. In thesame way, the sample coefficients describe the signal coming from thematerial inside the sample but covered by the membrane. One of ourobjectives in the following will be to relate experimentally themeasured coefficient to the classical theoretical coefficientscharacteristic of the material itself (e.g. water, gold, etc.).

Let us consider two adjacent points that give signals SA and SB. Thematerial contrast between them is defined as:C _(AB)=(S _(A) −S _(B))/S _(A)  (2)

(assuming S_(A)>S_(B)).

There are two basic ways to modify the signal S. One is to multiply itby a constant (amplification). The other is to add a positive ornegative constant to the signal (this is the “black level”). Thecontribution of the membrane to the signal is the same everywhere. Itsthickness and its composition are very well defined. So that neithercomposition contrast nor topographic contrast appear between points Aand B because of the membrane.

Contribution n is thus a constant that can be removed by an appropriatechoice of the black level. Note, though, that noise on the order of pnwhere n is the number of electrons scattered from the membrane, mayinterfere with the measurement, but we ignore it for the moment,returning to it below. With this choice, the contrast between points Aand B is greatly simplified:C _(AB)=(ηS _(A) −ηS _(B))/ηS _(A)  (3)

The material contrast is related only to the difference in the number ofbackscattering events occurring in the materials located below themembrane on points A and B.

Coming back to the contribution m, it generates a noise that maycompletely destroy the image if it is of the order of the amplitude ofthe signal generated by events inside the sample. For that reason, wehave to study the membrane characteristics and quantify that portion ofthe signal that is related to the membrane only and was suppressed inthe expression of the contrast (equation 3) by an appropriate choice ofthe black level.

C. Passage of Electrons Through the Membrane

We proceed now to quantify experimentally the contribution of themembrane to the signal. We do this in terms of BSE and SE emitted fromits surface as a function of the energy. The method we employ is tomeasure the total current crossing the sample in different situations,then subtracting to obtain the losses into BSE and SE. We measure thecurrent with no obstruction to estimate the full current that the SEMprovides at the sample, and then measure the effect of the membrane onthis current. Finally, we measure the current doing through the membraneto a given material, in out case gold and water.

For this purpose we used four different samples and an assembly thatallows the different samples to be inserted simultaneously inside themicroscope (of the four samples we had, three could be insertedtogether, so we repeated the experiment with overlapping trios andidentical conditions). For different energies, the beam was positionedsuccessively on each sample and the current between the ground and thesample was measured.

Sample One is just a Faraday Cup [Goldstein et al., Scanning ElectronMicroscopy and X-ray Microanalysis, pp. 65. Plenum press, New York,London, 1992], used to measure the beam current I_(beam) by collectingall the charge of the beam (a negligible quantum of charge can escapebackward). It is constructed of a bulk of carbon connected to ground,the a cavity (2 mm diameter, 3 mm deep) at its top: closed by a Ni platewith an aperture of 10_m diameter in the middle. The beam enters throughthe aperture, hits the bottom of the carbon cavity, so that the mainbeam and practically all scattered electrons are collected.

Sample Two is identical in design except that the aperture is coveredwith a membrane. We call this measurement I_(membrane).

Sample Three consists of a pure gold sample connected to ground andcovered by a membrane. The gold was first melted so that its surface wassmooth enough to obtain large areas where a ‘direct’ contact with themembrane was achieved. Finally, Sample Four is our experimental chamber,containing water and sealed by a membrane. Samples Three and Four definetwo values for I_(material)+I_(membrane).

The difference of currents measured on Samples One and Two (normalizedby that of Sample One) gives the percentage of electrons emitted byinteraction with the membrane. This measures the contribution of thesecondary and backscattering coefficients of the membrane:[(I _(beam) −I _(membrane))/I _(beam)]=η_(m)+δ_(m):

The difference of the beam current (Sample One) from the currentsmeasured on Samples Three or Four gives the percentage of electronsemitted from the sample that reach the outside. These include electronsthat interacted with both the membrane and the material (either gold orwater respectively):[(I _(beam) −I _(membrane+material)) I_(beam)]=(η_(m)+δ_(m))+η_(s)(1+Δ_(m)):

The subscript s refers to the material, here gold or water.

For energies below 5 keV, the gold and water curves are seen tosuperpose on the membrane curve. This means that the beam is notenergetic enough to both reach the sample and send BSE back to thedetector.

For energies above 5 keV, the three curves differ in their slope. Agrowing portion of the signal detected is due to interaction with thematerial considered. As the energy increases, this different increases:the material component of the signal becomes dominant.

For the water sample and incident electrons energy of 10 keV,beam-membrane interactions contribute 50% of the charge emission. Theircontribution decreases to 35% at 15 keV.

In the case of gold, because of its high atomic number the proportion isdramatically smaller. At 10 keV, only 4% of the charges emitted arecharacteristic of the membrane. At 15 keV, the membrane contributionfalls to 2%.

At this stage we can subtract the membrane component from the totalelectron emission, and obtain the material contribution to the signal.This includes only electrons that are scattered by their interactionwith the material of interest.

At low energies the electrons of the beam do not reach the samples andthe curves should be zero. At very, high energy, the curves shouldfollow the behavior of the backscattering coefficients of the materials,since the membrane becomes practically transparent. This is indeed whatwe observe on the two curves. The decreasing slope of the water curvefollows the theoretical behavior of the backscattering coefficient, ascalculated from Hunger and Kuchler's derivation [Phys. Status. Solidi A56, K45, 1979; see also Goldstein op cit, p. 95]. The increasing slopeof the gold curve also follows directly the calculated backscatteringcoefficient evolution with energy (as derived from the same expression).

By normalizing these curves by the backscattering coefficient of therespective materials (with their appropriate energy dependence), we getcurves where only a low material dependence remains.

The results for water indicate a backscattering coefficient that islower by about 25% than the theoretical curve predicted by Hunger andKuchler. We are not sure what the origin of this difference is, butbecause our sample is completely enclosed with minimal chance ofcontamination, we believe that our experimentally determined value isthe cleanest result available at this time for this quantity.

Though we have not measured this directly, the properties of thematerial below the membrane clearly affects also the energy spectra ofBSE that are emitted. The energies range from 0 to E₀ where E₀ is theenergy of the incident electrons. A light material (e.g. carbon) has adistribution that is approximately centered on the value E₀/2 and issymmetric [Bishop in Proceedings of the 4^(th) Intl. Conf. X-ray Opticsand Microanalysis, edited by Hermann (Paris, 1966); see also Goldsteinop cit., p. 100]. A heavy material (e.g. gold) has an energydistribution of BSE that is strongly asymmetric, with a peak closer toE₀. This difference induces two effects. First, the ratio of BSE thatare able to cross back through the membrane to the total BSE produced ishigher for heavy materials than for light ones. The membrane is actuallyacting to filter out the low energy BSE, thus enhancing artificially thecontrast between light and heavy elements. Second, the difference inenergy spectra of BSE can also influence the SE emission Δm since lowenergy BSE have a higher probability to generate SE on their way out.

For other materials of atomic number between that of water and gold, thecorresponding curve will lie between the two extremes shown. Wesummarize our conclusions from the results up to now as follows:

a) For energies below 4-5 keV, no signal from the sample can bedetected. This threshold can be decreased by using thinner membranes.

b) The intermediate region of energies is interesting for severalreasons: first, the membrane filtering effect on BSE of low energyenhances the contrast between heavy and light materials. Second, theemission of SE in this regime is important and it is possible to imageeasily with the SE detector. The peak on the gold curve reaches a valuehigher than 1, demonstrating a high emission of SE.

c) For very high energies, the charges emitted approach thetheoretically predicted BSE coefficients. The reason is that at higherelectron energies (for both electrons of the beam and the backscatteringelectrons) the probability for BSE to cross the membrane is higher, andthe probability to SE on their way out is lower. The secondary emissionrelated to Δm decreases and therefore, as observed experimentally, itbecomes easier to image through the BSE detector.

The membrane filtering effect on BSEs becomes negligible at highenergies. In the expression of the contrast (equation 3), thebackscattering coefficients can be replaced by the backscatteringcoefficients of the materials considered (water, gold, carbon etc.). Theenergy at which the plateau is reached shows the minimum energy abovewhich this approximation becomes valid. For the membrane exemplifiedherein, this value is around 15 keV.

D. Deducing a Minimum Probe Current for Observation of a Given MaterialContrast

As soon as a small difference of composition (difference in the meanatomic numbers) exist between two points, it is theoretically possibleto observe it. The question is how many electrons need to impinge onthose points to make that observation.

Such an estimate is useful for several reasons. First, the currentchosen influences the resolution: a high current causes the beam to beless focussed. This is more of a problem when the electron source of themicroscope is a tungsten filament and is a less sensitive issue for afield emission microscope. Second, the electron source can only producea limited number of charges, and third, high radiation doses can damagethe specimen.

We are interested, in general, in samples containing liquids such asemulsions, biological specimens, polymer solutions, etc., which in mostcases prove to be low contrast samples.

A somewhat arbitrary but useful criterion for sufficient visibility isthe “Rose Criterion” [Goldstein op cit., pp. 215]. It states that, inorder to observe a difference between two points A and B, the differenceof signal amplitude between those points must be higher than 5 times thelevel of noise:(S _(A) −S _(B))>5N.

Measuring the number of electrons that enter the detector when the beamis at point A at different scans, we get a distribution whose standarddeviation is given by

-   -   ˜η^(1/2) and centered on a mean value_n. The signal is thus        proportional to η, and the noise to η^(1/2). The signal to noise        ratio is S/N˜η^(1/2) and the minimum level of material contrast        that can be observed is Cmin=5/η^(1/2).

Thus the mean number of electrons that must be collected in order toobserve a specific material contrast C is given, in a general way, byη>(5/C)². The corresponding current (at the detector) isI_(S)>(q/t)(5/C)², where q is the electron charge and t the time thebeam is on each point, t can also be expressed as t=T/n_(p) where T isthe scan time and n_(p), the number of pixels per frame. For ahigh-quality image, there are typically n_(p)=106pixels. Measuring I_(S)in pico-Amperes and T in seconds, we find that the current required atthe detector to obtain a given material contrast C is:I _(S)>4/TC ²  (4)

Finally, the signal current I_(S) and the beam current I_(B) are relatedthrough

IS=ηS(ε_(BS)+ε_(SE)Δ_(m))I_(B). An SE detector like ai Everhart-Thornleydetector is very efficient

with good rejection of BSE [Goldstein op cit.]. The BSE detector doesnot collect SE and, because it has usually a small signature, has onlymedium collection efficiency. We found experimentally that at energiesaround 10 keV, both images in BSE and SE mode appear with the samecontrast while at higher energies the BSE mode is better. Taking areasonable estimate of the collection efficiency at 25% we get that thebeam current required to obtain a contrast C is [19] (also in [Goldsteinpp. 217]):I _(B)>16/_(ηs) TC ²(pA)  (5)

At high energies, the contrast C is simply related by equation 3 to thebackscattering coefficients of the materials below the membranes. TableI shows results of the calculation of the minimal current required foran observation of biological specimens such as a cell, an oil/wateremulsion and a gold particle in water.

The four elements C, H, N and O make up nearly 99% of the weight of abiological specimen like a cell. The cell is composed of 70% water and29% organic compounds. About 50%, of the atoms in these compounds are Hatoms, 24% are C, 24% are O, and 1% are N atoms [20]. Given thesevalues, the mean atomic number of a typical organic molecule is 6.72.That of water is 7.22, and for a living cell, it is 7.07. Calculation ofthe corresponding backscattering coefficient at 20 keV give 0.075 forwater, 0.073 for a cell. Thus, a cell surrounded by an aqueous mediumpresents a low contrast C=2.5% and furthermore the backscatteringcoefficients are low. This calculation shows that a minimal current inthe range of nano-Amperes is required for a typical scan of 100s.

material Z η contrast to water I (pA) water 7.22 0.075 — — Cells 7.070.073 0.027 2900 Oil 5.8 0.055 0.267 30 Gold 79 0.78 0.90 0.25

TABLE I. Evaluation of the minimum current needed for an observation ofdifferent samples containing water. I is calculated from equation 5. Thevalues for η are calculated from the expression of Hunger and Kuchler atan energy E=20 keV, using the means atomic number values Z indicated.The scanning time T=100s is typical of a meticulous slow scan.

An oil/water emulsion may be more easily observable and presents acontrast ten times higher: C=26.7%. The minimal current falls in thatcase to a few tens of pico-Amperes. For a gold particle immersed inwater, the contrast is very high (C=90%).

Imaging it requires only a fraction of a pico-Ampere (table I).

A biological sample such as a cell needs a high current to be imaged ina reasonable integration time. One can advantageously use labeling withheavy markers such as colloidal gold particles to decorate the cell.Emulsions of oil/water can be observed directly in standard conditionsof current and integration time.

At low energies (10 keV and under) the influence of the membrane becomesimportant. As a consequence, low contrast samples need more and morecurrent to be imaged.

E. Evaluation of the Resolution of the Technique

As in any electron microscope measurement, the question of resolutionultimately boils down to the existence of scattering atoms that have ahigh electron content. In the absence of strongly scattering atoms thebeam will reach far into the sample, widening and losing resolution asit does so, before it undergoes enough interactions to be returned. In amaterial with high electron density, the beam travels only a shortdistance before being returned or absorbed, so the scattering volume issmall and the resolution high.

To see the effect of the membrane on the resolution we need to evaluatethe effective signal producing volume, characterized by a diameterd_(eff) [Goldstein op cit., pp. 162]:d _(eff)=(d ² _(B) +d ² _(m) +d ² _(BSE))^(1/2)  (6)where d_(B) represents the diameter of the beam entering the membrane.As the beam crosses the membrane, some scattering occurs, which isparticularly important at low energy and tends to be negligible at highenergy. d_(m) represents the widening of the beam as a result of itsinteraction with the membrane—a beam with zero initial width d_(B)=0will emerge from the membrane with diameter d_(m).

The distance d_(BSE) is the mean diameter of the volume covered byelectrons in the sample before returning as BSE, d_(BSE) can be found inthe literature [Goldstein op cit, pp. 105]and is related to theKanaya-Okayama (KO) range. Rescaling all characteristic lengths by theKO range removes a large part of the material dependence, leaving only asmall atomic number Z dependence. In the case of a gold sample 90% ofthe BSE originate in a volume of diameter d_(BSE)=0.3 R_(KO). For carbonthe same relation gives dBSE=0.6 R_(KO).

The KO range gives the typical size of the volume of interaction of theelectrons inside the material. It is equivalent to the radius of acircle centered on the surface at the beam impact point whosecircumference encompasses the limiting envelope of the interactionvolume:R _(KO)=0:0276(A/Z)Z ^(0.11) E ₀ ^(1.67)/ρ  (7)R_(ko) is expressed in μm, E₀ is the incident beam energy in keV , A isthe atomic weieht in g/mole. ρ is the density in g/cm³ and Z is theatomic number.

It is non-trivial to calculate the theoretical resolution in the generalcase of three different layers (carbon membrane-material). However, inthe two limiting cases of materials with very low or very high atomicnumbers such an estimate is possible.

A. Resolution of low contrast samples at low energy.

Light materials such as water, oil or the material composing themembrane, are close enough in atomic number so that a simple estimationwould be to consider them as the same. Then, an estimate of theresolution is simplyd _(eff)=(d ² _(B) +d ² _(BSE))^(1/2)  (8)where d_(BSE) is calculated in a mean light material. This evaluationmakes sense as long as d_(BSE) is larger than the membrane thickness.

The diameter of the beam d_(B) depends on the electron source used inthe microscope. A Field Emission source produces a beam of 1 mm size inthe best conditions (low current, high energy), while in the mostdifficult conditions, the size is around 10 nm. For a tungsten filamentmicroscope, the diameter of the beam may vary from 10 to 100 mm

Thus, the resolutions obtained in the case of light samples are on theorder of 200 nm. At 10 keV , a low current and a Field Emission source,the estimation gives

d_(eff)=170 nm while at 20 keV, it goes up to d_(eff)=500 nm.

Experimentally, we found that it is hard to obtain a resolution betterthan the membrane thickness. When lowering the energy, the resolutionimproves because the volume sampled below the membrane diminishes. Butin practice. as the energy is decreased, the contrast decreases in atradeoff. Less electrons are backscattered from the sample. jnd theratio of the sample signal to membrane signal decreases. Thus, the reallimit of resolution is given by the minimum contrast that can bedetected. lEone can reduce the thickness of rhe membrane then theminimtim energy to image the sample will decrease and the resolutionwill improve significantly.

This estimate of the resolution indicates why at this stage for lowcontrast samples like an oil in water emulsion, our technique does norimprove much on optical microscopy. It can be improved by technicaladvances such as a thinner membrane. However, the situation completelychanges when looking at gold particles in water, or more generally atsmall objects made of heavy material and immersed in a light material.

B. Resolution of high contrast samples at high energy.

The case of gold particles inside water is very interesting. Theresolution obtained experimentally at high energy is in the range of fewtens of nanometers. The higher the energy the better is the resolutionobtained. Beads of actual diameter 40 nm appear to be around 50 nmdiameter at 30 keV, over an order of magnitude smaller than the volumeof interaction of BSE (given by dBSE). This demonstrates that theresolution is not related to the range of in interaction. In this case,the resolution is linked to the diameter of the beam when reaching thedepth of the particles.

At high energies the beam can diffuse inside the water if there is nobead to intercept it, and the volume sampled by the BSE is deep and wide(one to few microns). The signal that the BSE generate is the result ofintegrating over a very large volume and for this reason, does not varysignificantly when the beam is scanned. Furthermore, at highmagnification, when the image size itself is on the order of the size ofthe BSE sampled volume this deep BSE signal is a constant that can beremoved by an appropriate choice of the blacks level. On the other hand,when the electron beam crosses a heavy material bead a significant partof the beam can be intercepted and immediately a lot more BSE areemitted (high backscattering coefficient). Thus, the resolution obtainedis dependent oil the spatial extension of the electron beam at the depthof the bead.

A bead that is located just below the membrane is imaged with the bestresolution. The deeper the particle is inside the sample the lover isthe resolution at which it can be imaged.

The spreading of the beam is a problem that is well adapted to solutionby Monte-Carlo simulations. We used a code devised by D. C. Joy [MonteCarlo Modeling for electron Microscopy and Microanalysis (OxfordUniversity Press, New York, London 1995)] to perform such a calculation.Input values for the simulation are the Polyimide stoichiometric formulaC₂₂O₅H₁₀N₂, the mean atomic number 6:4 and the mean atomic weight 9.8g/mol. The density of the material is 1.4 g/cc.

At 20 keV the limit of resolution is estimated at 60 nm below themembrane. At 30 keV, the beam diameter calculated is 35 nm a valuecompatible with the resolution obtained experimentally when the beadsare in contact with the membrane.

The calculations show that if using a membrane twice thinner, imagingwith 10 nm resolution is possible at high energies.

For beads located deeper inside the sample, one must take into accountthe spreading of the beam due the layer of water in between. Anapproximation of the resolution is given by d_(eff)=√(d² _(beam)+d²_(membrane)+d² _(water)). d_(water) calculated from Monte-Carlosimulations.

With the membrane used and at 30 keV, a gold particle located at 100 nmbelow the membrane can be imaged with a resolution of 45 mm. A bead 200nm deep can be imaged with a resolution of 80 nm with the same energy.

F. Sampling Depth.

For completeness, it is of interest to evaluate how deep a heavyparticle can be detected inside the sample.

In a light material, almost 50% of the BSEs that escape reach a depthequal to 0.2R_(KO) before returning to the surface [Goldstein op cit.,pp. 105]. The numerical coefficient 0.2 is for carbon, but it should bevery similar for water and the material composing the membrane since thecoefficient evolves very slowly with atomic number.

Taking that value as our estimated sampling depth, at 6 keV the samplingdepth is around 150 nm. This value is compatible with our observationthat imaging was hardly possible below this energy with our 145 nm thickmembrane.

In water, the sampling depth is approximately 0.4 μm at 10 keV, 1.2 μmat 20 keV and 3.1 μm at 35 keV, which is the maximum energy available onmany scanning electron microscopes. These values give an estimate of therange of depths within which a gold particle can be detected insidewater.

G. Estimation of Pressure Inside Chamber.

The following is a simple calculation of the pressure inside a wetchamber. The calculation mostly refers to the setup, where the chamberis assumed filled with water with only very little air inside (say,small bubbles).

To simplify the calculation the following assumptions are considered:

-   -   1. We start with a closed chamber, which is tilled mostly with        water at atmospheric pressure. We do have a small volume of air        inside. V_(a) (measured in atmospheric pressure), which is small        compared to the volume expansion. V, after putting the chamber        in vacuum.    -   2. The membrane is modeled as a piston held by a spring with a        spring constant k. This modeling, is probably adequate since it        only assumes a linear relation between the force on the membrane        and the membrane's displacement, X. This effective constant is        easily estimated experimentally. The surface area of the        membrane (piston) is denoted by S.    -   3. Water is assumed to be incompressible. Once the chamber is        put in vacuum the piston shifts until equilibrium is reached.        The expanded volume, V_(e), is determined by equilibrium        condition and is filled by air and water vapor. The water vapor        pressure. P_(H2O), is determined by temperature only and is        independent of the volume (see Kittley's Thermal Physics). Both        air and water vapor are assumed to be ideal gases.

The number of water vapor molecules is given by:

$\begin{matrix}{N_{H2O} = \frac{P_{H2O}V_{e}}{RT}} & (1)\end{matrix}$where T is the temperature.

The total pressure in the chamber after insertion to vacuum is:

$\begin{matrix}{P_{tot} = {\frac{N_{tot}{RT}}{V_{e}} = \frac{( {N_{a} + N_{H2O}} )\mspace{11mu}{RT}}{V_{e}}}} & (2)\end{matrix}$where

$N_{a} = \frac{P_{anm}V_{a}}{RT}$is the number of air (nitrogen) molecules. In (1) and (2) we usedassumption 1 that V_(e)>V_(a).

Another equation for the pressure is given by the equilibrium condition:P_(tot)S=KX  (3)using V_(e)=XS we get an equation for the volume. V_(e):

$\begin{matrix}{V_{e} = \frac{P_{tot}S^{2}}{k}} & (4)\end{matrix}$

Putting (4) in (1) and (2) one gets after some manipulation an equationfor P_(tot) that depends on initial parameters:

$\begin{matrix}{P_{tot}^{2} = {\frac{P_{anm}V_{a}k}{S^{2}} + {P_{H2O}P_{tot}}}} & (5)\end{matrix}$

This equation can be transformed to (by dividing by the last term):

$\begin{matrix}{{\frac{P_{tot}}{P_{H2O}} = {1 \div \frac{P_{anm}V_{a}}{P_{H2O}V_{e}}}}{{or}\mspace{14mu}{to}}} & (6) \\{\frac{P_{tot}}{P_{anm}} = {\frac{P_{H2O}}{P_{anm}} + \frac{V_{a}}{V_{e}}}} & (7)\end{matrix}$

Equation (7) is useful for the estimation of P_(tot). The first term onthe right is about 0.025. If the ratio on the second term is muchsmaller than this value then air bubbles can be neglected and thepressure in the chamber is the water vapor pressure. If it is largerthen this ratio gives the fraction of the total pressure fromatmospheric pressure. Note that this equation was derived with thecondition that V_(e)>V_(a). If V_(a)>V_(e) then the pressure will stayclose to atmospheric pressure and the water vapor pressure isirrelevant.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference is now made to FIG. 1, which is a generalized diagram showinga longitudinal cross-section of the sample area of a prior art SEM 10. Aprimary electron beam 12 travels through a vacuum path to reach a sample14. It strikes the sample 14 to form a stream of backscattered electrons16, which strike a backscattered electron detector iS. In addition tobackscattered electrons, secondary electrons 20 emitted by the sampleare accelerated towards a secondary electron detector 22.

The mean free path of an electron through the atmosphere is very smalland thus the entire electron path, from an emitter via a sample to adetector is in a vacuum, making it impossible to study wet samples orin-vivo processes.

Reference is now made to FIG. 2 which is a generalized diagram showing alongitudinal cross-section of a scanning electron microscope accordingto a first aspect of the present invention. Parts that are the same asthose shown in the previous figure are given the same reference numeralsand are not described again, except as necessary for an understanding ofthe present embodiment.

In FIG. 2 a primary electron beam 12 travels through a vacuum chamber 30towards a sample 32 which is placed within a chamber 34. Sealed withinthe chamber 34 is an atmosphere suitable for the retention of wet andliving tissue, in which atmosphere in-vivo processes are able to takeplace. The most suitable environment will comprise an aqueous medium.

In order to seal the chamber from the vacuum chamber, a barrier 36,typically a membrane, is stretched across an aperture defined by a ring38. The ring 38 is designed to hold the membrane 36 at its ends in anairtight fashion as will be shown in greater detail in connection withFIG. 3 below. The sample 32 is preferably placed close up to themembrane 36 so that the electron beam does not have to travel throughthe atmosphere within the chamber in order to reach the sample. In apreferred embodiment, the sample 32 is in actual physical contact withthe membrane and in the case of living tissue, may be thrown on themembrane or attached thereto using known methods, as will be exemplifiedhereinbelow.

Preferably, the chamber 34 comprises a relief outlet 40, which will bediscussed in detail in connection with FIG. 3 below. It furthercomprises an inlet 41 and outlet 42 for fluids that maintain theappropriate conditions for the specimen. For example, it is possible toutilize the inlet 41 and outlet 42 to change the chemical environment ofthe specimen being studied. This feature is particularly useful in thestudy of drugs, where it is possible to watch the dynamic reaction of acell as the concentration of a given drug is changed.

The membrane 36 may be a foil or a film and should preferably be able towithstand pressure Gradients of up to one atmosphere whilst at the sametime being transparent to an electron beam. In one exemplary embodiment,a layer of polyimide substantially 1500 Å thick is used as it canwithstand the pressure gradient and is transparent to electron beamshaving energies in the region of 3 keV and above. Thinner membranes aretransparent to 2 keV. More generally the thicknesses may lie within arange of 200-5000 Å in order to withstand the necessary pressuregradient and at the same time be transparent to electrons at the beamenergies available. Preferred materials are polyimide, for example ofmolecular formula C₁₂H₁₂N₂O, polyamide, polyamide-imide, polyethylene,polypyrrole and additional conducting polymers, parlodion, collodion,silicon dioxide, silicon monoxide, carbon and trademarked materialsincluding Kapton™, FormVar™, Vinylec™, Pioloform™ and ButVar™. Amembrane composed of polyimide as described above allows for the passageof the primary beam and the resulting backscattered electrons 16.

Secondary electrons (SEs) generally have lower energies than thosediscussed above and would normally tend to be absorbed in the water.Thus SEs that are emitted from dire sample are not normally detected.However, in a preferred embodiment, the SE signal is enhanced by coatingthe membrane with one or more layers 37, of material having a highsecondary electron yield. Typical materials include lithium fluoride.

As will be appreciated, the SEs are detected by the SE detector 22 andthus serve to enhance the overall signal.

In a preferred embodiment, the diameter of the exposed area of themembrane 36 within the aperture is substantially 1-3 mm. The membrane istypically supported on a grid, each opening within the grid having amaximum diameter of substantially 0.1 mm. The grid enhances the abilityof the membrane to withstand a pressure gradient. The grid isschematically shown in FIG. 3.

The membrane preferably permits the maintenance of normal atmosphericconditions within the chamber 34, and thus permits the inspection of arange of samples that includes living cells, organelles or partsthereof, and proteins. It further allows the inspection of surfactants,colloids, oil particles, emulsions, and polymeric or micellar solutions.In the pharmaceutical industry the chamber can, for example, be used toexamine dynamic properties of drugs, such as swelling, dissolution anddisintegration id even the resistance of cells to certain drugs. In thetextile industry the clamber may be used or investigating the wettingand drying of wool, cotton and synthetic fibers. Other fields to whichthe invention is applicable may include petroleum, food, geology,microelectronics, paper coating and materials science.

The chamber 34 itself can be filled with a gas or a liquid as desiredand the samples can be monitored at the molecular level due to thenanometer range resolution of the electron beam. The resolution loss dueto the membrane is negligible since there are very few scattering eventswhich occur therein. When the chamber is filled with gas, a pressurerelief device, preferably comprising a spring 43 and an additionalmembrane 44, may be provided in the chamber to prevent the membrane 36from bursting.

The chamber 34, in accordance with embodiments of the present inventionmay be incorporated into standard SEMs, as discussed below in respect ofFIG. 3. The chamber 34 is compatible with standard specimen holders orspecimen mounts. The specimen holder is designed according to thedimensions of standard commercial specimen holders so that it can beincorporated with ease into the specimen chamber, that is, the enclosureof the microscope for placement of the specimen holder.

Although, in the above, the chamber has been described as containingatmospheric pressure, this is not necessary for all samples and incertain cases lower pressures may be found to be suitable, hencereducing the pressure gradient across the membrane 36.

A difficulty in using an electron beam to observe a living cell or anin-vivo In process is that the electron beam itself damages the sample.Peak damage to DNA, for example, occurs at 4 keV. The choice of 3keV/1500 Å polyimide membrane is safely below the peak damage level.

The damage level can farther be optimized by finding an energy levelwhich minimizes damage to the individual sample.

Reference is now made to FIG. 3, which is a simplified slightly explodedcross sectional diagram of a further embodiment of the chamber of FIG.2, showing greater detail. A chamber 50, preferably of a size which fitsinto a conventional sample holder, comprises a sample holding assembly51, which in turn comprises a body member 52 enclosing a sample region54 having an open surface 55. In the example of FIG. 3 the body member52 is a standard SEM specimen mount. The sample region is enclosed by amembrane 56 of the type described above which is fitted over the exposedside of the sample region 54. A grid 59 is situated above the membraneand separated therefrom by a spacer 57. A closing member 58 fits overthe body member 52 in such a way as to grip the membrane 56 around itsedges. The closing member is attached to the body vial threaded screws62 which fit into corresponding holes in the enclosing members 58, whichholes are continuous with threaded holes 60 in the body member 52. Inorder to provide hermetic sealing of the sample region 54, an O-ringseal 66 is fitted in the body, member 52 around the sample region 54.

A sample 64 is shown in close proximity with the membrane 56.Preferably, it should be either in actual contact with the membrane orwithin nanometers thereof so that the electron beam does not have totravel through any significant quantity of atmosphere.

When the sample region is sealed by tightening of the screws 62 theremay be a build-up of pressure within the sample region. There is thusprovided a pressure-relief assembly 68 allowing for escape of excessfluid. The assembly 68 comprises a stem 70 enclosing a channel 72.Channel 72 connects the sample region 54 with a further opening 74 atthe far end of the stein 70. Around the stein 70 is a slidingcylindrical closing member 76 comprising an upper part 77 which fitsover a lower part 79 to press it inwardly against a second O-ring seal78. The closing member 76 is preferably used to seal the opening 74 onlyafter the sample region itself has been seated, thus allowing a path forthe excess fluid of the sample-holding assembly 51.

Inserts 73 allow for insertion into a sample holder (not shown).

Reference is now made to FIG. 4, which is an embodiment of the chamberof FIGS. 2 and 3 adapted for additional sampling by optical means. InFIG. 4, a chamber 80 is produced as part of a light guide 82. A membrane84, held by closing member 86, seals the chamber 80 from the vacuum. Areflective coating 90 preferably coats one end of the light guide 82,the other end leading in the direction of arrow 92 to a photomultipliertube (not shown). A sample 94 is marked with an electro-luminescent(fluorescent) marker which is excited by the electron beam. The light isdeflected in the direction of arrow 92 to be amplified by thephotomultiplier tube (not shown). Thus it is possible to detect singlephotons, and there is preferably provided a means for optical detectionat the nano-scale resolution of SEM.

Reference is now made to FIG. 5 which is an electron micrograph showinga wet specimen inspection using the embodiment shown FIG. 3. The sampleis an oil-in-water emulsion as used in a previous experiment todemonstrate ESEM contrast, described by Matthews (Proceedings of theInstitute of Physics. EMAG99, page 95, Sheffield, 1999), the contents ofwhich are hereby incorporated by reference. The electron micrograph wastaken with a JEOL 8400 SEM having low resolution, and a scale marker isshown. The resolution obtained using a membrane in accordance with thepresent invention is virtually the same as the resolution level achievedusing the SEM in the conventional manner without a membrane. Goodcontrast is obtained and the overall results are sufficient for wetspecimen inspection.

It is appreciated that various features of the invention which are, forclarity, described in the contexts of separate embodiments mar also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention which are, for brevity, described in thecontext of a single embodiment may also be provided separately or in anysuitable subcombination.

DETAILED DESCRIPTION OF PREFERRED METHODS OF THE INVENTION

Ideally, a technology for imaging wet samples including but not limitedto viable unfixed cells should deliver the following capabilities:

-   -   1. Imaging Properties:        -   Resolution of at least 100 nm, preferably in the range of            1-20 nanometers        -   High signal to noise and signal to background ratios    -   2. Physiological Environment of the Cell:        -   Ability to work in the wet environment of the wet unfixed            cell        -   Minimal radiation damage (minimal interference to imaging,            minimal effect on the biologically relevance of the results)    -   3. Throughputs:        -   Minimal sample preparation steps.        -   Automation Strategy

The current invention is based on a Scanning Electron Microscope, whichwe modify to meet the specifications outlined above.

Minimal Sample Preparation Steps

Classical SEM procedures requires coating of the sample to prevent itscharging. Here the aqueous electrolyte solution (medium) surrounding thecells is enough to prevent charging. Thus, sample preparation Goes notrequire coatings and allows working with living cells. The fact thatsample preparation does not require fixation, de-hydration or coating,drastically shortens and simplifies sample preparation procedurescompared to conventional SEM, and significantly enhances the quality ofthe results. Here is a sample preparation procedure as a non-limitingexample. This example is suitable for labeling extracellular cellcomponents, like the extracellular portion of membrane proteins. Otherlabeling techniques including labeling of intracellular cell components.The described in the examples section below. Labeling cells is done bythe regular immunolabeling procedures.

Our results demonstrate that this wet SEM technique is suitable for bothfixed and livid cells, and for both adherent and non-adherent cells.Furthermore, these results demonstrate that while labeling is needed tohave single molecule detection, cells may be imaged without labeling.

Automation Strategy.

The current invention also solves the low throughput of laboratory SEMs.It utilizes for biological samples the SEM automation developed for thesemiconductors industry for wafer-inspection. This idea of utilizing inbiology the SEM automation developed for wafer-inspection is describedat length in copending PCT application, entitled “Method ofidentification and quantification of biological molecules and apparatustherefore” (PCT/IL01/00764) There, the samples are dry, allowing forstraightforward usage of a Wafer Inspection Scanning Electron Microscope(WISEM) or WISEM-like microscope. This idea could not be applied tobiological samples obtained in their natural aqueous environment,because preparation of such samples to be compatible with the vacuumrequirements of conventional electron microscopy, including WISEM,impose drastic and complicated steps that feasibly prevent automation.The current invention enables imaging of aqueous samples, without theneed for these problematic sample preparation steps. Thus, allowing theutilization in biology the SEM automation developed for thesemiconductors industry for wafer-inspection. This utilization willgreatly reduce the cost of instrumentation required to implement themethod.

The automation strategy is a set of guidelines describing the proceduresto be used in an automated imaging cell samples with the WET SEMtechnology. In a preferred embodiment of the invention the automationprocedure will include: A ‘cell chip’ containing a large number ofisolated chambers where each chamber contains a sample prepared for wetSEM imaging. Each such subdivision (isolated chamber) contains apartition membrane on which cell samples (which may be identical ordifferent) are grown.

Standard automated fluid control can be used in the preparation of thesamples.

Once the cell chip is ready for imagine it can be taken to an automatedoptical imaging (or alternatively low magnification SEM scan) usingstandard optical microscopy or special setup, like optical fiber etc.,integrated into the SEM. This scan will provide an overview image of thecells in each chamber, their position and any other information relevantfor the experiment. The information on the cells in this scan will bedetected automatically using specifically developed algorithms.

The partition membrane is mounted on a metal grid for mechanicalstability. The grid supplies a convenient, extremely stable referencefor the coordinates of every image.

In the case where an optical scan is performed outside the SEM, the cellchip is transferred to the SEM chamber for the SEMart imaging. Alignmentalgorithms will be used to match between the coordinate systems of theoptical microscope and the SEM.

Once these two coordinate systems are matched the SEM will imagespecific regions of interest (ROI) chosen automatically from the opticalscan (or the low magnification SEM scan). Such ROI can include aspecific region within a cell where important biological activity takesplace or a region that is related to a specific sublocalization in thecell or a region which provides a statistical representation of otherregions in the cell. The gold colloids are to be imaged in each suchregion.

Such ROI can have different size hence requiring different magnificationof the SEM. Note that there is a trade off between the field of viewimaged and the size of labeling that can be observed. Hence, differentsize of labeling will be required for imaging at different field ofview. Specifically, for an image of a whole single cell a large field ofview is required. Such a large field of view means low magnification andability to observe only large gold colloids (depending on the resolutionof the scan). Alternatively, many small field of view images taken athigher magnification can be combined to provide a high resolutionpicture of the whole cell.

SEM images are taken automatically. This includes automated positioningof the SEM stage, automated focus and astigmatic corrections, automatedbrightness and contrast.

Images taken by the SEM may be analyzed automatically using imageanalysis algorithms. This Includes identification, counting andcomparison of the position and size of gold colloids observed.

Other signals call also bee measured in the SEM imaging. These includesignals from cathodoluminescent markers (as exemplified hereinbelow) orX-ray emission from the sample which can provide analytical chemicalanalysis of the samples.

While one cell chip is scanned with SEM another can be scannedoptically.

Very High Resolution Optical Imaging in the SEM

A very interesting option is to use the narrow electron beam to excitelight emission in the sample. Such light emission is calledcathodoluminiscence (CL) and has been studied on dehydrated samples inthe past (reviewed by Hough PVC, Scanning electron microscopy I, 257,1977). This will enable detection of light emission with a resolutionmuch better than the theoretical limit in light microscopy. Thissolution combines the high resolution of SEM imaging with the familiarfluorescent labeling. This option is lightly advantageous when wet andliving samples are imaged in the SEM. In this imaging system theelectron beam produces secondary electrons (SE) emission that excite CL(fluorescent) labeling molecules that, in turn, emit visible light. Thisemitted light is then collected in a collection pathway separate fromthe usual electron detection. Excitation by secondary electrons can beshown to be equivalent to broad band UV radiation (see Hough PVC,Scanning electron microscopy I, 257 1977). The advantages of this methodare numerous:

-   -   1. The combination of both fluorescent and gold labeling has        many biological advantages    -   2. Getting a high resolution image of labeled molecules can        provide new and important biological information.    -   3. Single photon detection is easily managed and assures        recovery of a sizable fraction of the photons emitted    -   4. The availability of DNA-encoded fluorescent proteins opens        new labeling opportunities for electron microscopy.    -   5. Unique probes such as quantum dots and fluorescent beads can        be used as markers in this technique providing versatility and        advantages unattainable in other techniques.

The embodiment of the wet-CL apparatus may be based on existing CLdetectors or may involve the development of novel collection geometriesand configurations. Existing detectors are, for example, those thatcollect light from the upper half plane (above the sample). In thismirror configuration an ellipsoidal mirror is placed above the sample,with a small hole in it to allow the passage of the electron beam. Lightis collected by the mirror and directed towards a window in themicroscope, beyond which a photon detector is situated. Such acollection configuration can be obtained commercially, for example fromGatan (previously Oxford Instruments). Below we detail two exemplaryconfigurations for collection in the lower half plane, in which photonsare collected below the sample, and directed by light guides to adetector outside the microscope.

The apparatus is made as follows: The sample chamber (made of stainlesssteel) is closed on the upper part by the partition membrane and on thebottom part by a small lens so that the membrane is located at the localplane of the lens. Thus the light emitted in the close proximity of themembrane and collected by the lens comes out collimated when leaving thetens. The sample chamber is put inside a stage where a prism redirectsthe light coming out of the lens at 90 degrees (i.e. horizontally).

The stage also contains a lens, which is used to refocus the light atthe entrance of an optical fiber. The optical fiber leads from the stageto a detector located outside the microscope. The detector used issingle photon module detection system. For each photon detected, theoutput signal is a TTL signal (0-5V square signal).

The output of the detector is connected to a computer card which countsthe number of TTL signals (photons) received. The computer card receivesalso several signals from the microscope: beginning of an imageacquisition; beginning of a new frame; beginning of a new line. Usingappropriate software specially designed, the image is reconstructed inreal-time from the collected data.

FIG. 6 shows the Sample Chamber with the different component parts a-Eassembled together. The individual parts are described in detail in FIG.7A-E

Assembling the Set-Up for Fluorescence Experiment

FIGS. 7(A-E) shows a first embodiment of an assembly used for thefluorescent experiments

Part A containing on the side a hole of several millimeters in diameter(not shown) is put up side down on the table. Part B is put insidetaking care to align screw holes of parts and B. Two o-rings areinserted in part B. The membrane is put in place. The membrane ismounted on a plastic support of 0.1 mm thickness. The diameter of theplastic support has to be chosen between the two diameters of theo-rings used. The hole of 3 mm diameter was previously preformed in themiddle of the plastic support, this is the region where the membrane isfree. A TEM grid is stuck on one side (the external one) of themembrane, in its middle. Part C is then inserted. It is important thatthe horizontal canal is aliened with the side hole of part A. The 6screws are inserted. The membrane, parts A, B and C are screwedtogether. The sample is then ready to be filled with water, or any otheraqueous medium. The liquid medium is deposited inside the free space inthe middle of part C (while it is still upside down). A thin pin (0.1 mmdiameter) is inserted through the hole practiced on the sides of part Aand C. The pin is inserted deep inside, until its extremity reaches thewet chamber. The other extremity of the pin goes outside. The lasto-ring is put in its place in part C. (At this stage the o-ring cannotbe put in place completely, because the pin is there, just below. Thisis normal and useful for the following steps). Part D (containing in themiddle the lens to collimate the light—not shown) is put in theassembly. The inner side of the lens is wet by the medium filling thechamber. Finally Part E is inserted. When turning Part E inside theassembly, the part D (with the lens) is pressed progressively on theo-ring (of Part C). Since the o-ring is not completely in place becauseof the pin, it does not isolate yet the chamber from the outside. Whenscrewing Part E, the volume of the wet chamber is decreased, thus themedium in excess can flow out below the o-ring, in close proximity tothe pin. Usually, some droplets flow out from the canal in Part C. Whenthe o-ring is pressed enough, and the lens is correctly placed, the pinis slowly pulled out. At that stage of the assembly, the o-ring in partB is free to occupy all the space. It comes to its natural positionwhere it is sealing the chamber. Thus, the chamber is finally completelysealed at that stage. This assembly (Parts A to E) is then turned to itsnormal position (up-side up) and inserted inside the microscope stagespecially designed for it.

FIG. 8, consisting of FIGS. 8A and 8B, shows a microscope stage suitablefor use with the assembly. The top cavity of the stage (2) is the recessinto which the Sample Chamber is inserted. Just below it is a prism. Theprism (1) is used to redirect the light in the direction of a hole. Inthis hole, a lens (not shown) focuses the light at the entrance of anoptical fiber.

In order to get better light collection efficiency an alternative setupwas designed where a direct coupling between the biological sample and alight guide is established. This alternative setup is shown in FIG. 9.It contains a vacuum tight chamber with a light going directly from thesample region to the single photon detector setup described above. Inthis drawing the housing (1), is attached to the support (2), and acover (3); using screws (6,7), within is situated a cavity disk (4),with the membrane (9), further connected to the light guide (5), sealedwith the o-rings (8).

Labeling Techniques.

The current invention is suitable for imaging cell components in eitherthe intracellular or the extracellular parts of a cell.

Different approaches can be considered to label intracellular parts of acell.

If the cells can be used after fixation, the common methods involvepermeabilization or extraction of the cell membrane.

An alternative approach, which is suitable to living cells, is to insertthe markers through a pinocytotic reaction. The cells can thereafter befixed or observed while they are alive. The pinocytotic reactionpreserves viability of the cell if it is performed under the appropriateconditions. It allows inserting molecules (fluorescent markers in mostof the applications known in the art) or nanoscale particles (asparticularly suitable for the present application). Some researchershave reported success in introducing beads on the micron scale insidecells also.

Permeabilization and extraction procedures are suitable for fixed cells.Several procedures can be adopted, depending if the operator want toremove completely the cell membrane or just create some pores.

Extraction and fixation means that the external membrane of the cell isfirst completely removed. Then, immediately after, the cell is fixed.This kind of procedure is good for visualizing structures in thecytoplasm, the nucleus or the cytoskeleton.

Fixation and permeabilization means the cells are fixed first andfurther, the cell membrane is made permeable.

Pinocytotic reaction is suitable for living cells. For example one canuse an agent such as the commercially available Influx (Molecular ProbesI-14402 to introduce particles inside the cells. The Influx cell-loadingtechnique is based on the osmotic lysis of pinocytotic vesicles.Briefly, compounds to be loaded are mixed at high concentration with ahypertonic medium, allowing the material to be carried into the cellsvia pinocytotic vesicles. The cells are then transferred to a hypotonicmedium, which results in the release of trapped material from thepinocytotic vesicles within the cells, filling the cytosol with thecompounds.

Following the protocols given by Molecular Probes we introducedColloidal Gold, hundred nanometer size gold particles, fluorescent beadson the micron scale, and quantum dots inside cells. The cells can befixed and gold or silver enhanced prior to observation. When using thismethod a certain percentage of the cells die. In most of the experimentsperformed, no more than 10-20% of the cells were lost.

Cell surface of living cells can be labeled with gold particles with lowdamage to the cells. An exemplary protocol developed for use inconjunction with the present invention utilizes streptavidin-biotinlabeling methods as are known in the art. The idea is to incubate thecells with biotin that will attach to a certain percentage of theproteins on the cell surface. Then, the cells are incubated withstreptavidin linked to gold particles that will attach to the biotin.The dosage of biotin for incubation must be carefully controlled, sincetoo much biotin on the membrane protein may perturb their activity andinduce cell death. Different studies have shown biotin can be used forcell tracking applications. Several studies dealt with red blood cells(e.g. Hoffmann-Fezer et al., Annals of Hematology, 74, 231-238, 1997:Ault, K. A. and C. Knowles, Exp Hematology, 23, 996-1001, 1995, andreferences therein). Biotin labels many cells with little or no changein their biology and permits both their detection and their recovery ifneeded.

Microcolumns. A further development that will increase throughput isparallel inspection with microcolumns. The microcolumns are miniaturescanning electron microscopes that are produced by integrated siliconprocesses. Due to their size, the microcolumns can operate in parallel;considerably reducing the scanning time and the bulkiness of a SEM basedsystem. Further details of the microcolumns are given in Feinerman andCrewe “Miniature Electron Optics”, Advances in Imaging and ElectronPhysics, Vol. 102, 187 (1998) as well as U.S. Pat. No. 5,122,663, thecontents of which are hereby incorporated by reference as if fullydisclosed herein. This idea of utilizing microcolumns-based electronmicroscopes in biological applications is described at length in thecopending PCT application, entitled “Method of identification andquantification of biological molecules and apparatus therefore”(PCT/IL01/00764).

The Following examples of the principles of the present invention areprovided solely for illustrative purposes intended to be construed in anon-limitative fashion.

EXAMPLES

Growing Cells on the Partition Membrane

The following are exemplary procedures for growing cells on the membranegiven as nonlimiting examples. The polyimide partition membranes areFirst coated with fibronectin (0.1 mg/ml) for 15 minutes. After washingwith PBS and culture medium, cells are plated in the usual ways atconcentrations of 800-1500 cells in 12-15 μl per chamber. After 24 hourscells are ready for manipulation/labeling. Most cell types can grow onthe partition membrane itself without the support of an additionalmatrix like fibronectin, but under such conditions they would be morelikely to be washed off during the extensive washing procedures. Theviability of the cells is not affected by any of these procedures.

The current invention is compatible with both adherent and non-adherentcells (as exemplified hereinbelow). For the purpose of imagingnon-adherent cells (lymphocytes for example), cells are first labeled insuspension, and then allowed to adhere for 30 minutes onto a partitionmembrane pre-treated with 0.4% poly-lysine for 1-3 hours.

Minimizing Radiation Damage

Currently we exemplify image dynamics of undisturbed processes only on ashort time scale (seconds). One kind of radiation damage that needs tobe taken care is the damage produced by tree radicals formed by theelectron beam hitting the water molecules.

One possible strategy against such damage is to include additives to themedium of the cells that would absorb free radicals produced by theradiation, thereby minimizing the radiation damage to the cells. Anyknown compounds suitable for radical scavenging may be used as long asit is non-toxic to the cells. One exemplary candidate for such anadditive is sucrose, which has been identified as a free radicalsabsorber. This solution is only possible with the current inventionsince conventional electron beam technology does not work in a wetenvironment.

Cell Labeling by Immunolabeling

Labeling cells is done by the regular immunolabeling procedures. Cellswere washed, and optionally fixed by 2% paraformaldehyde for 7 minutes.Following blocking with the appropriate serum, cells were incubated witha specific mono or polyclonal antibody for 1 hour. After washing, thesamples are incubated with a second antibody that recognizes the firstspecific antibody. This second antibody is linked to a 5, 10, 20 or 40nm gold cluster. The gold labeling may then be further amplified bysilver enhancement, using conditions carefully calibrated for eachcolloid.

Fixed Wet Cell Under the Partition Membrane

Growth Factor Receptors Labeled on Fixed A431 Cells and C2C12 MouseMyoblasts.

Cells were fixed with 2% paraformaldehyde and labeled with anti FGFreceptor monoclonal antibody (on C2C12 myoblasts) or anti-EGF monoclonalantibody (A431 cells) followed by anti mouse IgG linked to 20nm-diameter gold colloids. The cells were fixed, and silver enhancementwas used to increase the size of the labeling panicles. An XL30 ESEM wasused for imaging and the cells were examined with the backscatteredelectrons (BSE) mode. The FGF receptor gold labeling can be seen asbright spots on both cells shown in FIG. 10A. The EGF receptors arevisible on the A431 cells of FIGS. 10B-D. The fuzziness of the labelingaround the nucleus area is the result of topographic differences betweenthe gold particles. Gold labeling close to the partition membrane isclear and in focus, while the more distal labeling beyond and around thenucleus is fizzy. The fuzziness of the labels in the region of thenucleus is evidence for the fact that the labels in this region arefurther away from the partition membrane. This fuzziness actuallyprovides a tool for the determination of the distance of the labels fromthe plane of the partition membrane hence providing three-dimensionalinformation. Using image analysis algorithms that are capable ofdeconvoluting the effect of the additional medium between the partitionmembrane and the labels can provide a rather accurate estimation of thisdistance.

To achieve a higher resolution image of labels on the distal side of thecell (opposite to side attached to the membrane substratum) anotherchamber design can be applied. In such a design the cells are grown on aflat platform with only a thin layer of liquid covering them. Then thepartition membrane is placed on top of that platform covering the cells.In this way the top side of the cell is imaged and the cells are stillin wet environment.

Live Non-Labeled Adherent Cells Tinder the Membrane

Live C2C12 mouse myoblasts. Cells were plated on fibronectin in theirnormal culture medium. After 24 hours the cells were examined with theBSE mode. The live C2C12 cells (FIG. 11A-D) and CHO cells (FIG. 12) showclearly the outline of the cell and its nucleolus as well as somesub-nuclei organelles.

The IL-2 Receptor Alpha Labeled on Jurkat Human Lymphocyte

Cells were labeled with anti human IL-2 receptor monoclonal antibodyfollowed by anti mouse IgG linked to 20 nm-diameter gold colloids. Thecells were allowed to adhere onto a polylysine pre-coated partitionmembrane for 30 minutes and then examined with the BSE mode. FIG. 13shows in and out of focus gold labeling resulting from topographicdifferences between the gold particles as previously seen in the FGFreceptor labeling of fixed C2C12 (FIG. 10A), and EGF receptor labelingof A431 cells (FIGS. 10B-D).

Images Taken with this Cathodoluminescence (CL) Setup.

Even though Lie signal is rather weak (probably due to poor lightcollection efficiency), the cells are clearly observed. The cells areobserved even Without CL markers (i.e. some parts of the cell areautoluminescent). With CL markers and with improved collection toefficiency much higher resolution is expected.

Cathodoluminescence image of fixed cells NIH3T3. No luminescent markerswere inserted in the cells. Thus the light emitted results from the‘natural’ cathodoluminescence of the cells as shown in FIGS. 14A-C. Thecell width is about 20 microns.

Biotin-Based Labeling on NIH3T3 Cells:

Materials:

-   Sulfo-NHS-LC-biotin (Sigma B1022) (dissolve 10 mg/ml in DMSO and    keep at 4° C.).-   Streptavidin linked to gold particles of diameter 10 nm to 40 nm-   Phosphate buffered physiological saline (PBS) pre warmed at 37° C.-   Culture Medium for the cell line (pre warmed at culture temperature    for the cell line).-   Serum free medium pre warmed.    Solutions to Prepare:-   solution A: Sulfo-NHS-LC-biotin at 0.5 mg/ml in prewarmed PBS.    Solution has to be made just prior to use since degradation occurs    in water.-   solution B: Streptavidin gold dissolved (1/10 to 1/100) in PBS+serum    free medium (1 to 5 volume ratio).-   solution C: fill medium—PBS solution (1 to 5 volume ratio).    Procedure:    -   1. trypsinization of the cells.    -   2. centrifugation at 1250 t/min for 4 minutes.    -   3. remove supernatant and resuspend in 10 ml fresh medium    -   4. take 3 ml divided in two 1.5 ml eppendorf tubes.    -   5. centrifugation 1600 t/min for 3 minutes.    -   6. remove as much as possible the supernatant.    -   7. gently add PBS at 37 degrees to obtain 1.5 ml in the tubes        (washing). Resuspend cells.    -   8. centrifuge again for 3 minutes.    -   9. remove PBS and add biotin (solution A). Resuspend the cells        and mix well but gently.    -   10. incubate at 37 degrees for 20 minutes (shaking every 3 min)    -   11. centrifuge    -   12. remove supernatant and resuspend in 1.5 ml of fresh medium.    -   13. transfer to petri dishes or membranes and incubate for few        hours to overnight.    -   14. wash 1-2 times with PBS.    -   15. Incubate with Solution B for one hour and mix sometimes if        possible.    -   16. wash 1-2 times with PBS.    -   17. remove PBS and replace by solution C.    -   18. Proceed to observation in the electron microscope        Living NIHT3 cells. The cells surface proteins were biotinylated        and linked to Steptavidin attached to 40 nm gold particles, as        shown in FIG. 15.

It will be appreciated by persons skilled in the art that the presentinvention is not limited to what has been particularly shown anddescribed hereinabove. Rather, the scope of the present inventionincludes both combinations and subcombinations of the various featuresdescribed hereinabove as well as variations and modifications thereofwhich would occur to persons skilled in the art upon reading theforegoing description and which are not in the prior art.

1. A specimen enclosure assembly comprising: an enclosure structuredefining an enclosed specimen placement volume and defining an aperturecommunicating with said enclosed specimen placement volume; an electronbeam permeable and gas impermeable layer covering said aperture andsealing said specimen placement volume from a volume outside saidenclosure; and a pressure relief device communicating with said enclosedspecimen placement volume.
 2. A specimen enclosure assembly according toclaim 1 and wherein said layer is formed from a material selected fromthe group consisting of: polyimide, polyamide, polyamide-imide,polyethylene, polypyrrole, PARLODION, COLLODION, KAPTON, FORMVAR,VINYLEC, BUTVAR, PIOLOFORM, silicon dioxide, silicon monoxide andcarbon.
 3. A specimen enclosure assembly according to claim 1 and alsocomprising an apertured mechanical support for said layer, which definesopenings having at least one dimension of no less than 100 microns.
 4. Aspecimen enclosure assembly according to claim 1 and wherein saidenclosure structure and said layer are constructed and configured so asto ensure a specimen is engaged with said layer.
 5. A specimen enclosureassembly according to claim 1 and wherein said layer generally is gasimpermeable up to at least a pressure gradient of one atmosphere.
 6. Aspecimen enclosure assembly according to claim 1 and wherein saidenclosed specimen placement volume has dimensions all of which exceed 10microns and defines an aperture communicating with said enclosedspecimen placement volume.
 7. A specimen enclosure assembly according toclaim 1 and wherein said layer generally is permeable to electronshaving energies in excess of 2 KeV.
 8. A specimen enclosure assemblyaccording to claim 1 and also composing a light collector operative tocollect light generated from an interaction between electrons and aspecimen at a location within said enclosed specimen placement volume.9. A specimen enclosure assembly comprising: a specimen structuredefining an enclosed specimen placement volume; at least one electronbeam permeable, fluid impermeable, vacuum tolerant layer sealing saidspecimen placement volume from a volume outside said specimen structure;and a light collector operative to collect light generated from aninteraction between electrons and a specimen at a location within saidenclosed specimen placement volume.
 10. A specimen enclosure assemblyaccording to claim 9 and wherein said layer is formed from a materialselected from the group consisting of: polyamide, polymide,polyamide-imide, polyethylene, polypyrrole, PARLODION, COLLODION,KAPTON, FORMVAR, VINYLEC, BUTVAR, PIOLOFORM, silicon dioxide, siliconmonoxide and carbon.
 11. A specimen enclosure assembly according toclaim 9 and also comprising an apertured mechanical support for said atleast one layer, which defines openings having at least one dimension ofno less than 100 microns.
 12. A specimen enclosure assembly according toclaim 9 and wherein said at least one layer generally is gas impermeableup to at least a pressure gradient of one atmosphere.
 13. A specimenenclosure assembly according to claim 9 and wherein said enclosedspecimen placement volume has dimensions all of which exceed 10 micronsand defines an aperture communicating with said enclosed specimenplacement volume.
 14. A specimen enclosure assembly according to claim 9and wherein said layer generally is permeable to electrons havingenergies in excess of 2 KeV.
 15. A specimen enclosure assembly accordingto claim 9 and also comprising a pressure relief device communicatingwith said enclosed specimen placement volume.
 16. A method forperforming scanning electron microscopy comprising: providing a beam ofelectrons to a specimen region maintained at a pressure below the vaporpressure of water; placing a specimen in a specimen enclosure having anaperture and defining an enclosed specimen placement volume sealed atsaid aperture by an electron beam permeable, fluid impermeable, vacuumtolerant cover; placing said specimen enclosure in said beam ofelectrons; and detecting backscattered electrons from interaction ofsaid beam of electrons with said specimen.
 17. A method for performingscanning electron microscopy comprising: providing a beam of electronsto a specimen region maintained at a pressure below the vapor pressureof water; placing a specimen in a specimen enclosure assembly disposedin the specimen region at a pressure below the vapor pressure of waterand comprising: an enclosure structure defining an enclosed specimenplacement volume and defining an aperture communicating with saidenclosed specimen placement volume; an electron beam permeable and gasimpermeable layer covering said aperture and sealing said specimenplacement volume from a volume outside said enclosure, said enclosurestructure and said layer being constructed and configured so as topermit impingement of electron beams into said specimen placementvolume; placing said specimen enclosure assembly in said beam ofelectrons; and analyzing results of interactions of said beam ofelectrons with said specimen.
 18. A method for performing scanningelectron microscopy according to claim 17 and wherein analysis of saidresults of interactions of said beam of electrons with said specimenemploys X-rays.
 19. A method for performing scanning electron microscopyaccording to claim 17 and wherein analysis of said results ofinteractions of said beam of electrons with said specimen employs light.20. A method for performing scanning electron microscopy according toclaim 17 and wherein analysis of said results of interactions of saidbeam of electrons with said specimen employs electrons.